Fkbp52-tau interaction as a novel therapeutical target for treating the neurological disorders involving tau dysfunction

ABSTRACT

The invention relates generally to neuroprotection and repair in neurological disorders involving Tau dysfunction (including Alzheimer&#39;s disease). The invention describes AND INCLUDES a direct interaction between proteins FKBP52 and Tau. More particularly, the invention relates to a method for screening a drug for the prevention and treatment of neurological disorders involving Tau dysfunction comprising the following steps: a) determining the ability of a candidate compound, to modulate the interaction between a Tau polypeptide and a FKBP52 polypeptide and b) selecting positively the candidate compound that modulates said interaction. The present invention finally relates to diagnostic, prognostic, and monitoring assays of neurological disorders involving Tau dysfunction.

FIELD OF THE INVENTION

The invention relates generally to neuroprotection and repair inneurological disorders involving Tau dysfunction (including Alzheimer'sdisease). The invention describes a direct interaction between proteinsFKBP52 and Tau. The invention relates to a screening method formolecules acting on FKBP52-Tau interaction, in order to modulate thedetrimental effects of pathogenic Tau. The present invention finallyrelates to therapeutical diagnostic, prognostic, and monitoring assaysof neurological disorders involving Tau dysfunction.

BACKGROUND OF THE INVENTION

The Tau protein is a major Microtubule Associated Protein (MAP) widelyexpressed in the central nervous system, predominantly in neurons, whereit plays a key role in regulating microtubule dynamics, axonal transportand neurite outgrowth. Protein Tau occurs in the adult human brain undersix different isoforms generated by alternative splicing of exons 2, 3and 10 of a primary transcript of a unique gene located on chromosome17. The length of their sequences varies from 352 to 441 amino acids.Growing evidence suggests that aberrant assembly of “aggregated” Tau(natively unfolded protein) is a hallmark of a series of human cognitivediseases collectively referred to as tauopathies or neurologicaldisorders involving Tau dysfunction, which include Alzheimer's disease,Pick's disease, corticobasal degeneration, mild cognitive impairment,progressive supranuclear palsy, and frontotemporal dementia withchromosome 17-linked parkinsonism. Abnormalities of Tau such ashyperphosphorylation, mutation, truncation and the aggregation in“tangles” may be contributing factors to the pathogenic processes. Todate the role of Tau modifications in the induction of neurodegenerativediseases is not fully understood and deciphering the molecularmechanism(s) which control(s) Tau structure/function is therefore ofgreat interest and may help to find novel therapeutic approaches forthese diseases.

The instant invention is based on the discovery that Tau protein (all 6isoforms) binds specifically and directly the immunophilin FKBP52(FK506-Binding Protein). FKBPs are a family of ubiquitously expressedintracellular receptors for the powerful immunosuppressant drugs FK506and Rapamycin and therefore take place in the large group of proteinsknown as immunophilins. These proteins show a large distribution and areparticularly abundant in the nervous system suggesting novel andunexpected functions distinct from their immunomodulatory effects. Inaddition, neuroprotective effects of FK506 have been reported. Theselatter observations have provided new perspectives for the FKBP proteinfamily and a particular interest for FK506 and its non immunosuppressivederivative molecules as factors to devise new therapeutic assay fortreating lesions and diseases of the nervous system.

FKBP52, first identified and cloned as associated to steroid receptor(Lebeau et al., 1992), presents a modular organization. including fourindividual and functional domains (Callebaut et al., 1992). The FK506binding site of FKBP52 (domain I) localized in the N-terminal part ofthe protein (aa 1 to 149) contains a peptidyl prolyl-isomerase (PPIase)activity (Chambraud et al., 1993) characteristic of all immunophilinprotein family. While the second domain (aa 149 to 267) sharesstructural homology with domain I, the PPIase activity is residual andit does not bind FK506 (14,15); a noteworthy aspect of domain II is aconsensus ATP-GTP-binding sequence (16). The C-terminal domain, coveringdomains III and IV, includes a putative calmodulin binding site (Massolet al., 1992) and mediates, through its three tetratricopeptide repeat(TPR) (aa 273 to 389) the protein's interaction with HSP90 (Radanyi etal., 1994) which is also a component of steroid receptor complex(Catelli et al., 1980, Tai et al., 1986; Renoir et al., 1990). Inaddition the binding activity of FKBP52 with HSP90 is regulated bycasein kinase II which specifically phosphorylates FKBP52 (Myata et al.,1997).

Recently it has been reported that FKBP52 interacts with microtubulesand prevents tubulin polymerization (Chambraud et al., 2007). Resultsobtained so far suggest that this inhibition of tubulin polymerizationby FKBP52 may not only result from the sequestration of tubulin or froma modification of its structure, such a bending, by FKBP52, but thatanother important factor required in the assembling of tubulin intomicrotubules may be involved.

The inventors now postulate herein that the intervention of one or moremicrotubule stabilizing factor(s), such as microtubule-associatedproteins (MAPS), could explain the inhibition of tubulin polymerizationby FKBP52. Using classical biochemical and cellular approaches theyestablish solid foundations to the role of FKBP52 on Tau function anddiscover a direct and specific interaction between FKBP52 and Tau. ThisFKBP52-Tau interaction results in the modulation of the knownTau-mediated cellular functions such as: tubulin polymerization (FKBP52inhibits this function), Tau accumulation (FKBP52 inhibits thisaccumulation) and neurite outgrowth.

SUMMARY OF THE INVENTION

The inventors have discovered a direct and specific protein-proteininteraction between the immunophilin FKBP52 and the microtubuleassociated protein Tau (under all of its known isoforms,hyperphosphorylated or not). The invention establishes that theFKBP52-Tau interaction provides a new target that may be usedadvantageously for novel therapeutic approaches of neurologicaldisorders involving Tau dysfunction, and especially for Alzheimer'sdisease.

Therefore invention provides methods for identifying molecules thatmodulate the FKBP52-Tau interaction, therefore acting beneficially ontothe detrimental effects of the neurological disorders involving Taudysfunction.

The molecules identified via the method of the invention for theirability to modulate the interaction between Tau and FKBP52 are drugcandidates for the prevention and treatment of neurological disordersinvolving Tau dysfunction and in particular for the Alzheimer's disease.

The invention also establishes that the FKBP52-Tau interaction providesa biological marker, potentially involved in diagnostic, prognostic,clinical follow-ups.

DETAILED DESCRIPTION OF THE INVENTION Screening Methods of the Invention

A first object of the invention consists of a method for screening adrug for the prevention and treatment of neurological disordersinvolving Tau dysfunction comprising the following steps:

a) determining the ability of a candidate compound to modulate theinteraction between a Tau polypeptide and a FKBP52 polypeptide and

b) selecting positively the candidate compound that modulates saidinteraction.

As used herein the term “neurological disorders involving Taudysfunction” includes but is not limited to Alzheimer's disease,frontotemporal dementia, progressive supranuclear palsy, corticobasaldegeneration and frontotemporal lobar degeneration, also known as Pick'sdisease, as well as all neurological disorders which might, in thefuture, prove to involve Tau, such as the Parkinson disease forinstance.

In the context of the invention, the term “treating” or “treatment”, asused herein, means reversing, alleviating, or inhibiting the progress ofthe disorder or condition to which such term applies, or one or moresymptoms of such disorder or condition.

As used herein, the term “prevention” refers to preventing the diseaseor condition from occurring in a subject which has not yet beendiagnosed as having it.

As used herein the expression “a compound that modulates the interactionbetween FKBP52 and Tau” refers any compound having the capability toinhibit, decrease or enhance the interaction between protein Tau andprotein FKBP52.

At step a), any method suitable for the screening of protein-proteininteractions is suitable.

Whatever the embodiment of step a) of the screening method, the completeTau protein and the complete FKBP52 protein may be used as the bindingpartners. Alternatively, fragments of Tau protein and FKBP52 proteinthat include the site of interaction may be uses as the bindingpartners.

Therefore in one embodiment step a) of the screening method of theinvention consists of the following steps:

-   -   a1) bringing into contact the candidate compound to be tested        with a mixture of a first Tau polypeptide or a substantially        homologous or substantially similar amino acid sequence thereof        and (2) a second FKBP52 polypeptide or a substantially        homologous or substantially similar amino acid sequence thereof    -   a2) determining the ability of said candidate compound to        modulate the binding between said Tau polypeptide and said        second FKBP52 polypeptide.

The term “polypeptide” means herein a polymer of amino acids having nospecific length. Thus, peptides, oligopeptides and proteins are includedin the definition of “polypeptide” and these terms are usedinterchangeably throughout the specification, as well as in the claims.The term “polypeptide” does not exclude post-translational modificationsthat include but are not limited to phosphorylation, acetylation,glycosylation. and the like. Especially, the term includes allphosphoryaled forms of the polypeptide (e.g. all phosphorylated forms ofTau or FKBP52). Also encompassed by this definition of “polypeptide” arehomologs thereof.

Accordingly, the term “Tau polypeptide” refers to the Tau protein or afragment thereof that comprises the site of interaction with FKBP52protein. In the same manner, the term “FKBP52 polypeptide” refers to theFKBP52 protein or a fragment thereof that comprises the site ofinteraction with Tau protein.

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than 80%, preferably greater than85%, preferably greater than 90% of the amino acids are identical, orgreater than about 90%, preferably grater than 95%, are similar(functionally identical). The term “sequence identity” refers to theidentity between two peptides. Identity between sequences can bedetermined by comparing a position in each of the sequences which may bealigned for the purposes of comparison. When a position in the comparedsequences is occupied by the same base or amino acid, then the sequencesare identical at that position. A degree of sequence identity betweennucleic acid sequences is a function of the number of identicalnucleotides at positions shared by these sequences. A degree of identitybetween amino acid sequences is a function of the number of identicalamino acid sequences that are shared between these sequences. Todetermine the percent identity of two amino acids sequences or twonucleic acid sequences, the sequences are aligned for optimalcomparison. For example, gaps can be introduced in the sequence of afirst amino add sequence or a first nucleic acid sequence for optimalalignment with the second amino acid sequence or second nucleic acidsequence. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, the molecules are identical at that position. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences. In this comparison thesequences can be the same length or may be different in length. Optimalalignment of sequences for determining a comparison window may beconducted by the local homology algorithm of Smith and Waterman (J.Theor. Biol., 91 (2) pgs. 370-380 (1981), by the homology alignmentalgorithm of Needleman and Wunsch, J. Miol. Biol., 48(3) pgs. 443-453(1972), by the search for similarity via the method of Pearson andLipman, PNAS, USA, 85(5) pgs. 2444-2448 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetic ComputerGroup, 575, Science Drive, Madison, Wis.) or by inspection. The term“sequence similarity” means that amino acids can be modified whileretaining the same function. It is known that amino acids are classifiedaccording to the nature of their side groups and some amino adds such asthe basic amino acids can be interchanged for one another while theirbasic function is maintained.

In one embodiment the step a2) consists in generating physical valueswhich illustrate or not the ability of said candidate compound tomodulate the interaction between said first polypeptide and said secondpolypeptide and comparing said values with standard physical valuesobtained in the same assay performed in the absence of the saidcandidate compound. The “physical values” that are referred to above maybe of various kinds depending of the binding assay that is performed,but notably encompass light absorbance values, radioactive signals andintensity value of fluorescence signal. If after the comparison of thephysical values with the standard physical values, it is determined thatthe said candidate compound modulates the binding between said firstpolypeptide and said second polypeptide, then the candidate ispositively selected at step b).

The compounds that modulate the interaction between (i) the Taupolypeptide and (ii) the FKBP52 polypeptide encompass those compoundsthat bind either to the Tau polypeptide or to FKBP52 polypeptide,provided that the binding of the said compounds of interest thenmodulates the interaction between Tau and FKBP52.

Polypeptides of the invention may be produced by any technique known perse in the art, such as without limitation, any chemical, biological,genetic or enzymatic technique, either alone or in combination(s).

Knowing the amino acid sequence of the desired sequence, one skilled inthe art can readily produce said polypeptides, by standard techniquesfor production of polypeptides. For instance, they can be synthesizedusing well-known solid phase method, preferably using a commerciallyavailable peptide synthesis apparatus (such as that made by AppliedBiosystems, Foster City, Calif.) and following the manufacturer'sinstructions.

Alternatively, the polypeptides of the invention can be synthesized byrecombinant DNA techniques as is now well-known in the art. For example,these fragments can be obtained as DNA expression products afterincorporation of DNA sequences encoding the desired (poly)peptide intoexpression vectors and introduction of such vectors into suitableeukaryotic or prokaryotic hosts that will express the desiredpolypeptide, from which they can be later isolated using well-knowntechniques.

A wide variety of host/expression vector combinations are employed inexpressing the nucleic acids encoding for the polypeptides of thepresent invention. Useful expression vectors that can be used include,for example, segments of chromosomal, non-chromosomal and synthetic DNAsequences. Suitable vectors include, but are not limited to, derivativesof SV40 and pcDNA and known bacterial plasmids such as col EI, pCR1,pBR322, pMal-C2, pET, pGEX, pMB9 and derivatives thereof, plasmids suchas RP4, phage DNAs such as the numerous derivatives of phage I such asNM989, as well as other phage DNA such as M13 and filamentous singlestranded phage DNA; yeast plasmids such as the 2 microns plasmid orderivatives of the 2 microns plasmid, as well as centomeric andintegrative yeast shuttle vectors; vectors useful in eukaryotic cellssuch as vectors useful in insect or mammalian cells; vectors derivedfrom combinations of plasmids and phage DNAs, such as plasmids that havebeen modified to employ phage DNA or the expression control sequences;and the like.

Consequently, mammalian and typically human cells, as well as bacterial,yeast, fungi, insect, nematode and plant cells an used in the presentinvention and may be transfected by the nucleic acid or recombinantvector as defined herein. Examples of suitable cells include, but arenot limited to, VERO cells, HELA cells such as ATCC No. CCL2, CHO celllines such as ATCC No. CCL61, COS cells such as COS-7 cells and ATCC No.CRL 1650 cells, W138, BHK, HepG2, 3T3 such as ATCC No. CRL6361, A549,PC12, K562 cells, 293T cells, Sf9 cells such as ATCC No. CRL1711 and Cv1cells such as ATCC No. CCL70. Other suitable cells that can be used inthe present invention include, but are not limited to, prokaryotic hostcells strains such as Escherichia coli, (e.g., strain DH5-[alpha]),Bacillus subtilis, Salmonella typhimurium, or strains of the genera ofPseudomonas, Streptomyces and Staphylococcus. Further suitable cellsthat can be used in the present invention include yeast cells such asthose of Saccharomyces such as Saccharomyces cerevisiae.

In one embodiment, any Tau derived or FKBP52 polypeptide of theinvention is labelled with a detectable molecule for the screeningpurposes.

According to the invention, said detectable molecule may consist of anycompound or substance that is detectable by spectroscopic,photochemical, biochemical, immunochemical or chemical means. Forexample, useful detectable molecules include radioactive substance(including those comprising 32P, 25S, 3H, or 1251), fluorescent dyes(including 5-bromodesosyrudin, fluorescein, acetylaminofluorene ordigoxigenin), fluorescent proteins (including GFPs and YFPs), ordetectable proteins or peptides (including biotin, polyhistidine tailsor other antigen tags like the HA antigen, the FLAG antigen, the c-mycantigen and the DNP antigen).

According to the invention, the detectable molecule is located at, orbound to, an amino acid residue located outside the said amino acidsequence of interest, in order to minimise or prevent any artefact forthe binding between said polypeptides or between the candidate compoundand or any of said polypeptides.

In another particular embodiment, the polypeptides of the invention arefused with a GST tag (Glutathione S-transferase). In this embodiment,the GST moiety of the said fusion protein may be used as detectablemolecule. In the said fusion protein, the GST may be located either atthe N-terminal end or at the C-terminal end. The GST detectable moleculemay be detected when it is subsequently brought into contact with ananti-GST antibody, including with a labelled anti-GST antibody. Anti-GSTantibodies labelled with various detectable molecules are easilycommercially available.

In another particular embodiment, the polypeptides of the invention arefused with a poly-histidine tag. Said poly-histidine tag usuallycomprises at least four consecutive hisitidine residues and generally atleast six consecutive histidine residues. Such a polypeptide tag mayalso comprise up to 20 consecutive histidine residues. Saidpoly-histidine tag may be located either at the N-terminal end or at theC-terminal end In this embodiment, the poly-histidine tag may bedetected when it is subsequently brought into contact with ananti-poly-histidine antibody, including with a labelledanti-poly-histidine antibody. Anti-poly-histidine antibodies labelledwith various detectable molecules are easily commercially available.

In a further embodiment, the polypeptides of the invention are fusedwith a protein moiety consisting of either the DNA binding domain or theactivator domain of a transcription factor. Said protein moiety domainof transcription may be located either at the N-terminal end or at theC-terminal end. Such a DNA binding domain may consist of the well-knownDNA binding domain of LexA protein originating form E. Coli. Moreoversaid activator domain of a transcription factor may consist of theactivator domain of the well-known Gal4 protein originating from yeast.

In one embodiment of the screening method according to the invention,the first Tau polypeptide and second FKBP52 polypeptide as describedabove, comprise a portion of a transcription factor. In said assay, thebinding together of the first and second portions generates a functionaltranscription factor that binds to a specific regulatory DNA sequence,which in turn induces expression of a reporter DNA sequence, saidexpression being further detected and/or measured. A positive detectionof the expression of said reporter DNA sequence means that an activetranscription factor is formed, due to the binding together of saidfirst Tau polypeptide and second FKBP52 polypeptide polypeptide.

Usually, in a two-hybrid assay, the first and second portion of atranscription factor consist respectively of (i) the DNA binding domainof a transcription factor and (ii) the activator domain of atranscription factor. In some embodiments, the DNA binding domain andthe activator domain both originate from the same naturally occurringtranscription factor. In some embodiments, the DNA binding domain andthe activator domain originate from distinct naturally occurringfactors, while, when bound together, these two portions form an activetranscription factor. The term “portion” when used herein fortranscription factor, encompass complete proteins involved in multiprotein transcription factors, as well as specific functional proteindomains of a complete transcription factor protein.

Therefore in one embodiment of the invention, step a) of the screeningmethod of the invention comprises the following steps:

(1) providing a host cell expressing:

-   -   a first fusion polypeptide between (i) a Tau polypeptide as        define above and (ii) a first protein portion of transcription        factor    -   a second fusion polypeptide between (i) a FKBP52 polypeptide as        defined above and (ii) a second portion of a transcription        factor

said transcription factor being active on DNA target regulatory sequencewhen the first and second protein portion are bound together and

said host cell also containing a nucleic acid comprising (i) aregulatory DNA sequence that may be activated by said activetranscription factor and (ii) a DNA report sequence that is operativelylinked to said regulatory sequence

(2) bringing said host cell provided at step 1) into contact with acandidate compound to be tested

(3) determining the expression level of said DNA reporter sequence

The expression level of said DNA reporter sequence that is determined atstep (3) above is compared with the expression of said DNA reportersequence when step (2) is omitted. A different expression level of saidDNA reporter sequence in the presence of the candidate compound meansthat the said candidate compound effectively modulates the bindingbetween the Tau polypeptide and the FKBP52 polypeptide and that saidcandidate compound may be positively selected a step b) of the screeningmethod.

Suitable host cells include, without limitation, prokaryotic cells (suchas bacteria) and eukaryotic cells (such as yeast cells, mammalian cells,insect cells, plant cells, etc.). However preferred host cell are yeastcells and more preferably a Saccharomyces cerevisiae cell or aSchizosaccharomyces pombe cell.

Similar systems of two-hybrid assays are well know in the art andtherefore can be used to perform the screening method according to theinvention (see. Fields et al. 1989; Vasavada et al. 1991; Fearon et al.1992; Dang et al., 1991, Chien et al. 1991, U.S. Pat. No. 5,283,173,U.S. Pat. No. 5,667,973, U.S. Pat. No. 5,468,614, U.S. Pat. No.5,525,490 and U.S. Pat. No. 5,637,463). For instance, as described inthese documents, the Gal4 activator domain can be used for performingthe screening method according to the invention. Gal4 consists of twophysically discrete modular domains, one acting as the DNA bindingdomain, the other one functioning as the transcription-activationdomain. The yeast expression system described in the foregoing documentstakes advantage of this property. The expression of a Gal1-LacZ reportergene under the control of a Gal4-activated promoter depends on thereconstitution of Gal4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A compete kit (MATCHMAKER,TM) for identifying protein-protein interactions is commerciallyavailable from Clontech.

So in one embodiment, a first Tau polypeptide as above defined is fusedto the DNA binding domain of Gal4 and the second FKBP52 polypeptide asabove defined is fused to the activation domain of Gal4.

The expression of said detectable marker gene may be assessed byquantifying the amount of the corresponding specific mRNA produced.However, usually the detectable marker gene sequence encodes fordetectable protein, so that the expression level of the said detectablemarker gene is assessed by quantifying the amount of the correspondingprotein produced. Techniques for quantifying the amount of mRNA orprotein are well known in the art. For example, the detectable markergene placed under the control of regulatory sequence may consist of theβ-galactosidase as above described.

In another one embodiment, step a) comprises a step of subjecting to agel migration assay the mixture of the first Tau polypeptide and thesecond FKBP52 polypeptide as above defined, with or without thecandidate compound to be tested and then measuring the binding of thesaid polypeptides altogether by performing a detection of the complexesformed between said polypeptides. The gel migration assay can be carriedout as known by the one skilled in the art.

Therefore in one embodiment of the invention, step a) of the screeningmethod of the invention comprises the following steps:

(1) providing a first Tau polypeptide and a second FKBP52 polypeptide asdefined above

(2) bringing into contact the candidate compound to be tested with saidpolypeptides

(3) performing a gel migration assay a suitable migration substrate withsaid polypeptides and said candidate compound as obtained at step (2)

(4) detecting and quantifying the complexes formed between saidpolypeptides on the migration assay as performed at step (3).

The presence or the amount of the complexes formed between thepolypeptides are then compared with the results obtained when the assayis performed in the absence of the candidate compound to be tested.

The detection of the complexes formed between the said two polypeptidesmay be easily performed by staining the migration gel with a suitabledye and then determining the protein bands corresponding to the proteinanalysed since the complexes formed between the first and the secondpolypeptides possess a specific apparent molecular weight. Staining ofproteins in gels may be done using any well known methods in the art.Suitable gels are well known in the art but it is preferred to use nondenaturant gels. In a general manner, western blotting assays are wellknown in the art and have been widely described (Rybicki et al., 1982;Towbin et al. 1979; Kurien et al. 2006).

In a particular embodiment, the protein bands corresponding to thepolypeptides submitted to the gel migration assay can be detected byspecific antibodies. It may used both antibodies directed against theTau polypeptides and antibodies specifically directed against the FKBP52polypeptides.

In another embodiment, the said two polypeptides are labelled with adetectable antigen as above described. Therefore, the proteins bands canbe detected by specific antibodies directed against said detectableantigen. Preferably, the detectable antigen conjugates to the Taupolypeptide is different from the antigen conjugated to the FKBP52polypeptide. For instance, the first Tau polypeptide can be fused to aGST detectable antigen and the second FKBP52 polypeptide can be fusedwith the HA antigen. Then the protein complexes formed between the twopolypeptides may be quantified and determined with antibodies directedagainst the GST and HA antigens respectively.

In another embodiment, step a) included the use of an optical biosensorsuch as described by Edwards et al. (1997) or also by Szabo et al.(1995). This technique allows the detection of interactions betweenmolecules in real time, without the need of labelled molecules. Thistechnique is indeed based on the surface plasmon resonance (SPR)phenomenon. Briefly, a first protein partner is attached to a surface(such as a carboxymethyl dextran matrix). Then the second proteinpartner is incubated with the previously immobilised first partner, inthe presence or absence of the candidate compound to be tested. Then thebinding including the binding level, or the absence of binding betweensaid protein partner is detected. For this purpose, a light beam isdirected towards the side of the surface area of the substrate that doesnot contain the sample to be tested and is reflected by said surface.The SPR phenomenon causes a decrease in the intensity of the reflectedlight with a combination of angle and wavelength. The binding of thefirst and second protein partner causes a change in the refraction indexon the substrate surface, which change is detected as a change in theSPR signal.

In another one embodiment of the invention, the screening methodincludes the use of affinity chromatography.

Candidate compounds for use in the screening method above can also beselected by any immunoaffinity chromatography technique using anychromatographic substrate onto which (i) the first Tau polypeptide or(ii) the second FKBP52 polypeptide as above defined, has previously beenimmobilised, according to techniques well known from the one skilled inthe art. Briefly, the Tau polypeptide or the FKBP52 polypeptide as abovedefined may be attached to a column using conventional techniquesincluding chemical coupling to a suitable column matrix such as agarose,Affi Gel®, or other matrices familiar to those of skill in the art. Insome embodiment of this method, the affinity column contains chimericproteins in which the Tau polypeptide or FKBP52 polypeptide as abovedefined, is fused to glutathion-s-transferase (GST). Then a candidatecompound is brought into contact with the chromatographic substrate ofthe affinity column previously, simultaneously or subsequently to theother polypeptide among the said first and second polypeptide. The afterwashing, the chromatography substrate is eluted and the collectedelution liquid is analysed by detection and/or quantification of thesaid later applied first or second polypeptide, so as to determine if,and/or to which extent, the candidate compound has modulated the bindingbetween (i) first Tau polypeptide and (ii) the second FKBP52polypeptide.

In another one embodiment of the screening method according to theinvention, the first Tau polypeptide and the second FKBP52 polypeptideas above defined are labelled with a fluorescent molecule or substrate.Therefore, the potential alteration effect of the candidate compound tobe tested on the binding between the first Tau polypeptide and thesecond FKBP52 polypeptide as above defined is determined by fluorescencequantification.

For example, the first Tau polypeptide and the second FKBP52 polypeptideas above defined may be fused with auto-fluorescent polypeptides, as GFPor YFPs as above described. The first Tau polypeptide and the secondFKBP52 polypeptide as above defined may also be labelled withfluorescent molecules that are suitable for performing fluorescencedetection and/or quantification for the binding between saidpolypeptides using fluorescence energy transfer (FRET) assay. The firstTau polypeptide and the second FKBP52 polypeptide as above defined maybe directly labelled with fluorescent molecules, by covalent chemicallinkage with the fluorescent molecule as GFP or YFP. The first Taupolypeptide and the second FKBP52 polypeptide as above defined may alsobe indirectly labelled with fluorescent molecules, for example, by noncovalent linkage between said polypeptides and said fluorescentmolecule. Actually, said first Tau polypeptide and second FKBP52polypeptide as above defined may be fused with a receptor or ligand andsaid fluorescent molecule may be fused with the corresponding ligand orreceptor, so that the fluorecent molecule can non-covalently bind tosaid first Tau polypeptide and second FKBP52 polypeptide. A suitablereceptor/ligand couple may be the biotin/streptavifin paired member ormay be selected among an antigen/antibody paired member. For example, apolypeptide according to the invention may be fused to a poly-histidinetail and the fluorescent molecule may be fused with an antibody directedagainst the poly-histidine tail.

As already specified, step a) of the screening method according to theinvention encompasses determination of the ability of the candidatecompound to modulate the interaction between the Tau polypeptide and theFKBP52 polypeptide as above defined by fluorescence assays using FRET.Thus, in a particular embodiment, the first Tau polypeptide as abovedefined is labelled with a first fluorophore substance and the secondFKBP52 polypeptide is labelled with a second fluorophore substance. Thefirst fluorophore substance may have a wavelength value that issubstantially equal to the excitation wavelength value of the secondfluorophore, whereby the bind of said first and second polypeptides isdetected by measuring the fluorescence signal intensity emitted at theemission wavelength of the second fluorophore substance. Alternatively,the second fluorophore substance may also have an emission wavelengthvalue of the first fluorophore, whereby the binding of said and secondpolypeptides is detected by measuring the fluorescence signal intensityemitted at the wavelength of the first fluorophore substance.

The fluorophores used may be of various suitable kinds, such as thewell-known lanthanide chelates. These chelates have been described ashaving chemical stability, long-lived fluorescence (greater than 0.1 mslifetime) after bioconjugation and significant energy-transfer inspecificity bioaffinity assay. Document U.S. Pat. No. 5,162,508discloses bipyridine cryptates. Polycarboxylate chelators with TEKEStype photosensitizers (EP0203047A1) and terpyridine typephotosensitizers (EP0649020A1) are known. Document WO96/00901 disclosesdiethylenetriaminepentaacetic acid (DPTA) chelates which usedcarbostyril as sensitizer. Additional DPT chelates with other sensitizerand other tracer metal are known for diagnostic or imaging uses (e.g.,EP0450742A1).

In a preferred embodiment, the fluorescence assay performed at step a)of the screening method consists of a Homogeneous Time ResolvedFluorescence (HTRF) assay, such as described in document WO 00/01663 orU.S. Pat. No. 6,740,756, the entire content of both documents beingherein incorporated by reference. HTRF is a TR-FRET based technologythat uses the principles of both TRF (time-resolved fluorescence) andFRET. More specifically, the one skilled in the are may use a HTRF assaybased on the time-resolved amplified cryptate emission (TRACE)technology as described in Leblanc et al. (2002). The HTRF donorfluorophore is Europium Cryptate, which has the long-lived emissions oflanthanides coupled with the stability of cryptate encapsulation. XL665,a modified allophycocyanin purified from red algae, is the HTRF primaryacceptor fluorophore. When these two fluorophores are brought togetherby a biomolecular interaction, a portion of the energy captured by theCryptate during excitation is released through fluorescence emission at620 nm, while the remaining energy is transfered to XL665. This energyis then released by XL665 as specific fluorescence at 665 nm. Light at665 nm is emitted only through FRET with Europium. Because EuropiumCryptate is always present in the assay, light at 620 nm is detectedeven when the biomolecular interaction does not bring XL665 within closeproximity.

Therefore in one embodiment, step a) of the screening method maytherefore comprises the steps consisting of:

(1) bringing into contact a pre-assay sample comprising:

-   -   a first Tau polypeptide fused to a first antigen,    -   a second FKBP52 polypeptide fused to a second antigen    -   a candidate compound to be tested

(2) adding to the said pre assay sample of step (2):

-   -   at least one antibody labelled with a European Cryptate which is        specifically directed against the first said antigen    -   at least one antibody labelled with XL665 directed against the        second said antigen

(3) illuminating the assay sample of step (2) at the excitationwavelength of the said European Cryptate

(4) detecting and/or quantifying the fluorescence signal emitted at theXL665 emission wavelength

(5) comparing the fluorescence signal obtained at step (4) to thefluorescence obtained wherein pre assay sample of step (1) is preparedin the absence of the candidate compound to be tested.

If at step (5) as above described, the intensity value of thefluorescence signal is different (lower or higher) than the intensityvalue of the fluorescence signal found when pre assay sample of step (1)is prepared in the absence of the candidate compound to be tested, thenthe candidate compound may be positively selected at step b) of thescreening method. Antibodies labelled with a European Cryptate orlabelled with XL665 can be directed against different antigens ofinterest including GST, poly-histidine tail, DNP, c-myx, HA antigen andFLAG which include. Such antibodies encompass those which arecommercially available from CisBio (Bedfors, Mass., USA), and notablythose referred to as 61GSTKLA or 61HISKLB respectively.

In a particular embodiment, the methods of the invention may furthercomprise a step consisting of determining the ability of the candidatecompound to modulate the interaction between a Amyloid Precursor Protein(APP) polypeptide and a FKBP52 polypeptide. Indeed a recent study havedemonstrated that FKBP52 formed stable complexes with APP through itsFK506 interacting domain. This study studies identify a novel role forFKBP52 in modulating toxicity of Abeta peptides (Sanokawa-Akakura R, CaoW, Allan K, Patel K, Ganesh A, Heiman G, Burke R, Kemp F W, Bogden J D,Camakaris J, Birge R B, Konsolaki M. Control of Alzheimer's amyloid betatoxicity by the high molecular weight immunophilin FKBP52 and copperhomeostasis in Drosophila. PLoS One. 2010 Jan. 13; 5(1):e8626.).Accordingly, compounds that modulate both interaction between Tau andFKBP52 and interaction between APP and FKBP52 may be useful for thetreatment of Alzheimer's disease.

In Cellulo Screening Methods of the Invention

The candidate compounds that have been positively selected at the end ofany one of the embodiments of the in vitro screening which has beendescribed previously in the present specification may be subjected tofurther selection steps in view of further assaying its properties onthe Tau mediated cellular functions (such as tubulin polymerisation),Tau accumulation, Tau aggregation and all the post-translational Taumodifications. For this purpose, the candidate compounds that have beenpositively selected with the general in vitro screening method as abovedescribed may be further selected for their ability modulates the Taumediated cellular functions, Tau accumulation, Tau aggregation and allthe post-translational Tau modifications.

Thus another aspect of the invention relates to a method for screening adrug for the prevention and treatment of neurological disordersinvolving Tau dysfunction, wherein said method comprises the steps of:

i) screening for compounds that modulate the interaction between the Tauand the FKBP52 proteins, by performing the in vitro screening methodaccording to any of claims 1 to 6 and

ii) screening the compounds positively selected at the end of step i)for their Tau mediated cellular functions, Tau accumulation, Tauaggregation and all the post-translational Tau modifications.

In certain preferred embodiments of the screening method above, step ii)of said screening method comprises the following steps:

(1) bringing into contact a cell with a compound that has beenpositively selected at the end of step i)

(2) determining the capacity of compound to modulate the Tau mediatedcellular functions, Tau accumulation, Tau aggregation and all thepost-translational Tau modifications

(3) comparing the Tau mediated cellular functions, Tau accumulation, Tauaggregation and all the post-translational Tau modifications determinedat step (2) with the Tau mediated cellular functions, Tau accumulation,Tau aggregation and all the post-translational Tau modifications thatare determined when step (1) is performed in the absence of the saidpositively selected compound.

For performing step (1), any cell may be suitable but neurons arepreferred.

Step (1) as above described may be performed by adding an amount of thecandidate compound to be tested to the culture medium. Usually, aplurality of culture samples are prepared, so as to add increasingamounts of the candidate compound to be tested in distinct culturesamples. Generally, at least one culture sample without candidatecompound is also prepared as a negative control for further comparison.Optionally, at least one culture sample with an already known agent thatmodulates the Tau mediated cellular functions, Tau accumulation, Tauaggregation and all the post-translational Tau modifications is alsoprepared as a positive control for standardisation of the method.

Therefore, step (3) may be performed by comparing the percentage ofcells wherein the Tau mediated cellular functions, Tau accumulation, Tauaggregation and all the post-translational Tau modifications aremodulated obtained for the cell cultures incubated with the candidatecompound to be tested with the percentage of cells wherein the Taumediated cellular functions, Tau accumulation, Tau aggregation and allthe post-translational Tau modifications are modulated obtained for thenegative control cell cultures without the said candidate compound.Illustratively, the efficiency of the candidate compound may be assessedby comparing (i) the percentage of cells wherein the Tau mediatedcellular functions, Tau accumulation, Tau aggregation and all thepost-translational Tau modifications are modulated measured in the cellcultures that were incubated therewith with (ii) the percentage of cellswherein the Tau mediated cellular functions, Tau accumulation, Tauaggregation and all the post-translational Tau modifications aremodulated measured in the supernatant of the cell cultures that wereincubated with the known agent that modulates the Tau mediated cellularfunctions, Tau accumulation, Tau aggregation and all thepost-translational Tau modifications. Further illustratively, theefficiency of the candidate compound may be assessed by determining forwhich amount of the candidate compound added to the cell cultures thepercentage of cells wherein the Tau mediated cellular functions, Tauaccumulation, Tau aggregation and all the post-translational Taumodifications are modulated is close or higher than the percentage ofcells wherein the Tau mediated cellular functions, Tau accumulation, Tauaggregation and all the post-translational Tau modifications aremodulated measured with the known agent that modulates the Tau mediatedcellular functions, Tau accumulation, Tau aggregation and all thepost-translational Tau modifications.

Candidate Compounds of the Invention

According to a one embodiment of the invention, the candidate compoundof may be selected from the group consisting of peptides,petptidomimetics, small organic molecules, antibodies, aptamers ornucleic acids. For example the candidate compound according to theinvention may be selected from a library of compounds previouslysynthesised, or a library of compounds for which the structure isdetermined in a database, or from a library of compounds that have beensynthesised de novo.

In a particular embodiment, the candidate compound may be selected formsmall organic molecules.

As used herein, the term “small organic molecule” refers to a moleculeof size comparable to those organic molecules generally sued inpharmaceuticals. The term excludes biological macromolecules (e.g.;proteins, nucleic acids, etc.); preferred small organic molecules rangein size up to 2000 da, and most preferably up to about 1000 Da.

The candidate compounds according to the invention may be selected frommolecules that neutralize the PPIase activity of FKBP52 by interferingwith its synthesis, translation and ligand/substrate/product-binding.

In a preferred embodiment, the molecules are selected from PPIaseligands of domain I of Protein FKBP52, preferably PPIase ligandspreventing, blocking or inhibiting the PPIase activity of domain I ofProtein FKBP52.

Advantageously, the molecules of the invention may prevent/inhibit/blockthe PPIase activity of domain I of Protein FKBP52 without inducingimmunosuppressive activity.

A first series of the molecules are derivatives of FK 506 that arelacking immunosuppresive activity. FK 506 is represented hereunder:

One particularly preferred family of molecules may consist of novelanalogues of the neurophilin compound represented below also named asVA-10367 (Vertex, Inc):

Other examples include derivatives of Rapamycin lacking theimmunosuppressant activity, such as Meridamycin as described in theInternational Patent Application Publication no WO2005084673. Otherderivatives of Rapamycin include those described in Ruan B. et al. (RuanB, Pong K, Jow F, Bowlby M, Crozier R A, Liu D, Liang S, Chen Y, MercadoM L, Feng X, Bennett F, von Schack D, McDonald L, Zaleska M M, Wood A,Reinhart P H, Magolda R L, Skotnicki J, Pangalos M N, Koehn F E, CarterG T, Abou-Gharbia M, Graziani E I. Binding of rapamycin analogs tocalcium channels and FKBP52 contributes to their neuroprotectiveactivities. Proc Natl Acad Sci USA. 2008 Jan. 8; 105(1):33-8. Epub 2007Dec. 27.) such as WYE-592 or ILS-290:

The molecules of the invention may be synthesized by any conventionalmethod well known to the skilled person.

In another particular embodiment, the candidate compounds according tothe invention may be antibodies o specifically directed to theinteraction site between protein Tau and protein FKBP52 or impacting theFKBP52-Tau interaction and/or its functional cellular functions.

The term “antibody” or “antibodies” relates to an antibody characterizedas being specifically directed to the interaction site of FKBP52 withTau, or any functional derivative thereof, with above mentionedantibodies being preferably monoclonal antibodies; or an antigen-bindingfragment thereof, of the F (ab′)2, or single chain Fv type, or any typeof recombinant antibody derived thereof. These antibodies of theinvention include specific polyclonal antisera prepared against theinteraction site of FKBP52 with Tau.

The antibodies of the invention can for instance be produced by anyhybridoma liable to be formed according to classical methods fromsplenic cells of an animal, particularly of a mouse or rat immunizedagainst the peptidic sequence involved in the interaction between Tauand FKBP52 or any functional derivative thereof, and of cells of amyeloma cell line, and to be selected by the ability of the hybridoma toproduce the monoclonal antibodies recognizing the peptidic sequenceinvolved in the interaction between Tau and FKBP52 or any functionalderivative thereof which have been initially used for the immunizationof the animals. The antibodies according to this embodiment of theinvention may be humanized versions of the mouse antibodies made bymeans of recombinant DNA technology, departing from the mouse and/orhuman genomic DNA sequences coding for H and L chains or from cDNAclones coding for H and L chains.

Alternatively the antibodies according to this embodiment of theinvention may be human antibodies. Such human antibodies are prepared,for instance, by means of human peripheral blood lymphocytes (PBL)repopulation of severe combined immune deficiency (SCID) mice asdescribed in PCT/EP 99/03605 or by using transgenic non-human animalscapable of producing human antibodies as described in U.S. Pat. No.5,545,806. Also fragments derived from these antibodies such as Fab, F(ab)′2 ands (“single chain variable fragment”), providing they haveretained the original binding properties, form part of the presentinvention. Such fragments are commonly generated by, for instance,enzymatic digestion of the antibodies with papain, pepsin, or otherproteases. It is well known to the person skilled in the art thatmonoclonal antibodies or fragments thereof, can be modified for varioususes. An appropriate label of the enzymatic, fluorescent, or radioactivetype can label the antibodies involved in the invention.

In another particular embodiment, the candidate compounds according tothe invention may be selected from aptamers. Aptamers are a class ofmolecule that represents an alternative to antibodies in term ofmolecular recognition. Aptamers are oligonucleotide or oligopeptidesequences with the capacity to recognize virtually any class of targetmolecules with high affinity and specificity. Such ligands may beisolated through Systematic Evolution of Ligands by EXponentialenrichment (SELEX) of a random sequence library, as described in TuerkC. and Gold L., 1990. The random sequence library is obtainable bycombinatorial chemical synthesis of DNA. In this library, each member isa linear oligomer, eventually chemically modified, of a unique sequence.Possible modifications, uses and advantages of this class of moleculeshave been reviewed in Jayasena S. D., 1999. Peptide aptamers consists ofa conformationally constrained antibody variable region displayed by aplatform protein, such as E. coli Thioredoxin A that are selected fromcombinatorial libraries by two hybrid methods (Colas et al., 1996).

In another embodiment, the candidate molecules may be selected frommolecules that block the synthesis of Protein FKBP52.

By synthesis is meant the transcription of the FKBP52 gene. Smallmolecules can bind on the promoter region of the FKBP52 and inhibitbinding of a transcription factor or these molecules can bind atranscription factor and inhibit binding to the FKBP52-promoter so thatthere is no expression of the FKBP52.

Also within the scope of the invention is the use of oligoribonucleotidesequences that include anti-sense RNA and DNA molecules and ribozymesthat function to inhibit the translation of FKBP52 mRNA. Anti-sense RNAand DNA molecules act to directly block the translation of mRNA bybinding to targeted mRNA and preventing protein translation. In regardto antisense DNA, oligodeoxyribonucleotides derived from the translationinitiation site. Ribozymes are enzymatic RNA molecules capable ofcatalysing the specific cleavage of RNA. The mechanism of ribozymeaction involves sequence specific hybridisation of the ribozyme moleculeto complementary target RNA, followed by an endonucleolytic

To inhibit the activity of the gene or the gene product of FKBP52custom-made techniques are available directed at three distinct types oftargets: DNA, RNA and protein; For example, the gene or gene product ofFKBP52 can be altered by homologous recombination, the expression of thegenetic code can be inhibited at the RNA levels by antisenseoligonucleotides, interfering RNA (RNAi) or ribozymes, and the proteinfunction can be altered by antibodies or drugs.

Another embodiment of present invention is the use of a molecule thatinhibits the expression of FKBP52 and preferably a molecule thatinhibits the expression of FKBP52 selected from the list consisting ofan antisense molecule, a RNAi and a ribozyme, for the manufacture of adrug to prevent or treat a disorder of Tau aggregation or deposition inneurofibrillary tangles in a subject in need thereof.

Methods of Treatment of the Invention

In a further aspect, the invention provides a method for the preventionand treatment of neurological disorders involving Tau dysfunctioncomprising administering a subject in need thereof with atherapeutically effective amount of a compound that modulate theinteraction between the Tau and the FKBP52 protein. Said compound may beidentified by the screening methods of the invention.

More particularly, the invention relates to a compound that modulatesthe interaction between the Tau and the FKBP52 protein for use in theprevention and treatment of neurological disorders involving Taudysfunction.

The methods of the invention are useful for the prevention and treatmentof neurological disorders involving Tau dysfunction includingAlzheimer's disease, frontotemporal dementia, progressive supranuclearpalsy, corticobasal degeneration and frontotemporal lobar degeneration,also known as Pick's disease, as well as all neurological disorderswhich might, in the future, prove to involve Tau, such as the Parkinsondisease for instance.

According to the invention, the term “patient” or “patient in needthereof”, is intended for a human or non-human mammal (e.g. dog, cat,horses . . . ) affected or likely to be affected with neurologicaldisorders involving Tau dysfunction.

By a “therapeutically effective amount” of the compound of the inventionis meant a sufficient amount of compound to treat neurological disordersinvolving Tau dysfunction, at a reasonable benefit/risk ratio applicableto any medical treatment. It will be understood, however, that the totaldaily usage of the compound of the invention and compositions of thepresent invention will be decided by the attending physician within thescope of sound medical judgment. The specific therapeutically effectivedose level for any particular patient will depend upon a variety offactors including the disorder being treated and the severity of thedisorder; activity of the specific compound employed; the specificcomposition employed, the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration, andrate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts. Forexample, it is well within the skill of the art to start doses of thecompound at levels lower than those required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved.

The compound that modulates the interaction between the Tau and theFKBP52 protein of the invention may be combined with pharmaceuticallyacceptable excipients. “Pharmaceutically” or “pharmaceuticallyacceptable” refers to molecular entities and compositions that do notproduce an adverse, allergic or other untoward reaction whenadministered to a mammal, especially a human, as appropriate. Apharmaceutically acceptable carrier or excipient refers to a non-toxicsolid, semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type.

In a further aspect, the invention provides a method for the preventionand treatment of Alzheimer's disease comprising administering a subjectin need thereof with a therapeutically effective amount of a compoundthat modulate both the interaction between the Tau and the FKBP52protein and the interaction between FKBP52 and APP.

Diagnostic Methods of the Invention

A further aspect of the invention pertains to diagnostic, prognostic,and monitoring assays.

Accordingly, in a particular embodiment, the invention relates to amethod of testing a subject thought to have or be predisposed to havinga neurological disorders involving Tau dysfunction, which comprises thestep of analyzing a sample of interest obtained from said subject formeasuring the level of the complex between FKBP52 protein and Tauprotein.

As used herein, the term “sample of interest” include encompasses avariety of sample types obtained from a subject and can be used in adiagnostic assay. Samples herein may be any type of sample, such as anycell samples, biological fluids including, blood, serum, urine, spinalfluid . . . or any biopsy sample obtained from a subject's tissue. In apreferred embodiment, the sample of interest is a spinal fluid sample.

In a particular embodiment, the methods of the invention comprisecontacting the sample of interest with a binding partner capable ofselectively interacting with the complex between FKBP52 protein and Tauprotein present in the sample of interest. In another particularembodiment, methods of the invention comprise may involve contacting thesample of interest with a binding partner capable of selectivelyinteracting with the FKBP52 protein and another binding partner capableof selectively interacting Tau protein present in the sample ofinterest.

The binding partners according to the invention may be an antibody thatmay be polyclonal or monoclonal, preferably monoclonal. In anotherembodiment, the binding partner may be an aptamer.

The binding partners of the invention such as antibodies or aptamers,may be labelled with a detectable molecule or substance, such as afluorescent molecule, a radioactive molecule or any others labels knownin the art. Labels are known in the art that generally provide (eitherdirectly or indirectly) a signal.

As used herein, the term “labelled”, with regard to the antibody, isintended to encompass direct labelling of the antibody or aptamer bycoupling (i.e., physically linking) a detectable substance, such as aradioactive agent or a fluorophore (e.g. fluorescein isothiocyanate(FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody oraptamer, as well as indirect labelling of the probe or antibody byreactivity with a detectable substance. An antibody or aptamer of theinvention may be labelled with a radioactive molecule by any methodknown in the art. For example radioactive molecules include but are notlimited radioactive atom for scintigraphic studies such as I123, I124,In111, Re186, Re188.

The aforementioned assays generally involve the bounding of the bindingpartner (ie. Antibody or aptamer) in a solid support. Solid supportswhich can be used in the practice of the invention include substratessuch as nitrocellulose (e. g., in membrane or microtiter well form);polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex(e.g., beads or microtiter plates); polyvinylidine fluoride; diazotizedpaper; nylon membranes; activated beads, magnetically responsive beads,and the like.

The level of the complex between FKBP52 protein and Tau protein may bemeasured by using standard immunodiagnostic techniques, includingimmunoassays such as competition, direct reaction, or sandwich typeassays. Such assays include, but are not limited to, agglutinationtests; enzyme-labelled and mediated immunoassays, such as ELISAs;biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis;immunoprecipitation.

More particularly, an ELISA method can be used, wherein the wells of amicrotiter plate are coated with a set of binding partners according tothe invention. A sample of interest containing or suspected ofcontaining the complex between FKBP52 protein and Tau protein is thenadded to the coated wells. After a period of incubation sufficient toallow the formation of antibody-antigen complexes, the plate(s) can bewashed to remove unbound moieties and a detectably labelled secondarybinding molecule added. The secondary binding molecule is allowed toreact with any captured sample marker protein, the plate washed and thepresence of the secondary binding molecule detected using methods wellknown in the art.

Alternatively an immunohistochemistry (IHC) method may be preferred. IHCspecifically provides a method of detecting targets in a tissue specimenin situ. The overall cellular integrity of the sample of interest istherefore maintained in IHC, thus allowing detection of both thepresence and location of the complex between FKBP52 protein and Tauprotein. Typically the sample is fixed with formalin, embedded inparaffin and cut into sections for staining and subsequent inspection bylight microscopy. Current methods of IHC use either direct labeling orsecondary antibody-based or hapten-based labeling. Examples of known IHCsystems include, for example, EnVision™ (DakoCytomation), Powervision®(Immunovision, Springdale, Ariz.), the NBA™ kit (Zymed LaboratoriesInc., South San Francisco, Calif.), HistoFine® (Nichirei Corp, Tokyo,Japan).

In particular embodiment, a tissue section may be mounted on a slide orother support after incubation with the binding partners of theinvention d. Then, microscopic inspections in the sample mounted on asuitable solid support may be performed. For the production ofphotomicrographs, sections comprising samples may be mounted on a glassslide or other planar support, to highlight by selective staining thepresence of the proteins of interest.

Therefore IHC samples may include, for instance: (a) preparationscomprising a cell sample or a tissue sample (b) fixed and embedded saidcells and (c) detecting the complex between FKBP52 protein and Tauprotein in said cells samples. In some embodiments, an IHC stainingprocedure may comprise steps such as: cutting and trimming tissue,fixation, dehydration, paraffin infiltration, cutting in thin sections,mounting onto glass slides, baking, deparaffination, rehydration,antigen retrieval, blocking steps, applying primary antibodies, washing,applying secondary antibodies (optionally coupled to a suitabledetectable label), washing, counter staining, and microscopicexamination.

In one embodiment, the method of the invention further may comprise astep of comparing the level of the complex between FKBP52 protein andTau protein with a predetermined threshold value. Said comparison isindicative whether the subject is thought to have or be predisposed tohaving a neurological disorders involving Tau dysfunction. Thepredetermined value may refer to the amount of the complex betweenFKBP52 protein and Tau protein in sample of interests obtained from thegeneral population or from a select population of subjects. For example,the select population may be comprised of apparently healthy subjects,such as individuals who have not previously had any sign or symptomsindicating the presence of neurological disorders involving Taudysfunction. In another example, the predetermined value may becomprised of subjects having established neurological disordersinvolving Tau dysfunction. The predetermined value can be a cut-offvalue, or a range. The predetermined value can be established based uponcomparative measurements between apparently healthy subjects andsubjects with established neurological disorders involving Taudysfunction.

Moreover, without wishing to be bound by theory, the inventors believethat aberrant expression or activity of FKBP52, and/or especially itsability to bind Tau thereby determine whether an individual is afflictedwith a neurological disorders involving Tau dysfunction, or is at riskof developing a neurological disorders involving Tau dysfunction.

Accordingly, in a particular embodiment, the invention relates to amethod of testing a subject thought to have or be predisposed to havinga neurological disorders involving Tau dysfunction, which comprises thestep of analyzing a sample of interest obtained from said subject fordetecting the presence of a mutation in the gene encoding for FKBP52protein and/or its associated promoter.

Typical techniques for detecting a mutation in the gene encoding forFKBP52 protein may include restriction fragment length polymorphism,hybridisation techniques, DNA sequencing, exonuclease resistance,microsequencing, solid phase extension using ddNTPs, extension insolution using ddNTPs, oligonucleotide assays, methods for detectingsingle nucleotide polymorphism such as dynamic allele-specifichybridisation, ligation chain reaction, mini-sequencing, DNA “chips”,allele-specific oligonucleotide hybridisation with single ordual-labelled probes merged with PCR or with molecular beacons, andothers.

In another particular embodiment, the invention relates to a method oftesting a subject thought to have or be predisposed to having aneurological disorders involving Tau dysfunction, which comprises thestep of analyzing a sample of interest obtained from said subject foranalyzing the expression of the gene encoding for FKBP52 protein.

Analyzing the expression of the gene encoding for FKBP52 protein may beassessed by any of a wide variety of well-known methods for detectingexpression of a transcribed nucleic acid or translated protein.

In a preferred embodiment, the expression of the gene encoding forFKBP52 protein is assessed by analyzing the expression of mRNAtranscript or mRNA precursors, such as nascent RNA, of said gene. Saidanalysis can be assessed by preparing mRNA/cDNA from cells in a sampleof interest from a subject, and hybridizing the mRNA/cDNA with areference polynucleotide. The prepared mRNA/cDNA can be used inhybridization or amplification assays that include, but are not limitedto, Southern or Northern analyses, polymerase chain reaction analyses,such as quantitative PCR (TaqMan), and probes arrays such as GeneChip™DNA Arrays (AFF-YMETRIX).

Advantageously, the analysis of the expression level of mRNA transcribedfrom the gene encoding for FKBP52 protein involves the process ofnucleic acid amplification, e. g., by RT-PCR (the experimentalembodiment set forth in U.S. Pat. No. 4,683,202), ligase chain reaction(BARANY, Proc. Natl. Acad. Sci. USA, vol. 88, p: 189-193, 1991), selfsustained sequence replication (GUATELLI et al., Proc. Natl. Acad. Sci.USA, vol. 57, p: 1874-1878, 1990), transcriptional amplification system(KWOH et al., 1989, Proc. Natl. Acad. Sci. USA, vol. 86, p: 1173-1177,1989), Q-Beta Replicase (LIZARD′ et al., Biol. Technology, vol. 6, p:1197, 1988), rolling circle replication (U.S. Pat. No. 5,854,033) or anyother nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill inthe art. These detection schemes are especially useful for the detectionof nucleic acid molecules if such molecules are present in very lownumbers. As used herein, amplification primers are defined as being apair of nucleic acid molecules that can anneal to 5′ or 3′ regions of agene (plus and minus strands, respectively, or vice-versa) and contain ashort region in between. In general, amplification primers are fromabout 10 to 30 nucleotides in length and flank a region from about 50 to200 nucleotides in length. Under appropriate conditions and withappropriate reagents, such primers permit the amplification of a nucleicacid molecule comprising the nucleotide sequence flanked by the primers.

In another particular embodiment, the invention relates to a method oftesting a subject thought to have or be predisposed to having aneurological disorders involving Tau dysfunction, which comprises thestep of analyzing a sample of interest obtained from said subject formeasuring the concentration of FKBP52 protein.

Measuring the concentration of the FKBP52 protein may be assessed byusing a binding partner as above described, and more particularly anantibody (e.g., a radio-labeled, chromophore-labeled,fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative(e.g., an antibody conjugate with a substrate or with the protein orligand of a protein of a protein/ligand pair (e.g.,biotin-streptavidin)), or an antibody fragment (e.g., a single-chainantibody, an isolated antibody hypervariable domain, etc.) which bindsspecifically to the FKBP52 protein.

Said analysis can be assessed by a variety of techniques well known fromone of skill in the art including, but not limited to, enzymeimmunoassay (EIA), radioimmunoassay (RIA), Western blot analysis andenzyme linked immunoabsorbant assay (RIA).

The methods of the invention may comprise comparing the level ofexpression of the gene encoding for FKBP52 protein or the concentrationof the FKBP52 protein in the sample of interest from a subject with apredetermined threshold value. The predetermined value may refer to theexpression level or concentration measured in sample of interestsobtained from the general population or from a select population ofsubjects. For example, the select population may be comprised ofapparently healthy subjects, such as individuals who have not previouslyhad any sign or symptoms indicating the presence of neurologicaldisorders involving Tau dysfunction. In another example, thepredetermined value may be comprised of subjects having establishedneurological disorders involving Tau dysfunction. The predeterminedvalue can be a cut-off value, or a range. The predetermined value can beestablished based upon comparative measurements between apparentlyhealthy subjects and subjects with established neurological disordersinvolving Tau dysfunction.

In another particular embodiment, the invention relates to a method oftesting a subject thought to have or be predisposed to having aneurological disorders involving Tau dysfunction, which comprises thestep of analyzing a sample of interest obtained from said subject fordetecting post-translational modifications of FKBP52 protein.

The post-translational modifications FKBP52 protein include but are notlimited to phosphorylation, acetylation, glycosylation. and the like.Detecting the post-translational modifications of the FKBP52 protein maybe assessed by using a binding partner specific for a post-translationalform of FKBP52 protein. As described above, the binding partner may bean antibody (e.g., a radio-labeled, chromophore-labeled,fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative(e.g., an antibody conjugate with a substrate or with the protein orligand of a protein of a protein/ligand pair (e.g.,biotin-streptavidin)), or an antibody fragment (e.g., a single-chainantibody, an isolated antibody hypervariable domain, etc.) which bindsspecifically to a specific form of the FKBP52 protein. Said analysis canbe assessed by a variety of techniques well known from one of skill inthe art including, but not limited to, enzyme immunoassay (EIA),radioimmunoassay (RIA), Western blot analysis and enzyme linkedimmunoabsorbant assay (RIA).

The methods as above described may then particularly suitable for theprognostic or predictive purpose to administer the subject with aprophylactic treatment prior to the onset of clinical signscharacterized by or associated with a neurological disorders involvingTau dysfunction. Clinical signs may include but are not limited to anysort of dysfunction of any CNS activity (such as motor/sensitiveactivities, sleep disturbances, depressive states, amnesic troubles,etc. . . . ). More particularly, the methods of the invention may bealso useful for monitoring the treatment of a subject affected with aneurological disorders involving Tau dysfunction.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1: FKBP52 in the brain. Twenty μg of cytosol proteins fromdifferent adult rat brain regions were analyzed by Western blottingusing anti-FKBP52 antibody 761. Actin served as the loading control.

FIGS. 2A-D: Association between FKBP52 and Tau proteins. A) GSTPull-down assay: Immunoblot (IB) for Tau showing the binding of solublemicrotubule extract proteins incubated with GST-tagged FKBP52, or GSTalone as control. B) Co-immunoprecipitation assay: A soluble microtubuleextract was subjected to immunoprecipitation (IP) with immunopurifiedanti-FKBP52 antibody, or pre-immune serum used as control. Thesupernatants (S) and precipitates (P) were immunoblotted with anti-Tauantibody (clone DC25). C) The ability of Tau proteins to bind FKBP52directly was monitored by dot blot assay. Different amounts ofrecombinant Tau (hT40) were spotted onto nitrocellulose membranes andthen assayed for bound FKBP52 (0.5 μg) using anti-FKBP52 antibody. 5 μgGST spotted onto nitrocellulose membrane were used as the control. D)Quantitation: 100% corresponds to 0.5 μg of FKBP52 loading before milksaturation, and 0% corresponds to 0.5 μg of FKBP52 loading after milksaturation. The background is defined as the signal when GST was loadedinstead of hT40. The level of FKBP52 captured by hT40 was calculatedafter substraction of background.

FIGS. 3A-C: Relevance of Tau phosphorylation for its interaction withFKBP52. A) Recombinant Tau (hT40), P-Tau and HP-Tau were analyzed bySDS-Page. Phosphorylation and hyperphosphorylation of HT40 resulted in amarked reduction in the gel mobility of recombinant Tau as shown on animmunoblot (IB), with anti-Tau antibody (clone DC25). B) Dot blot assaywith 2.2 μg of HP-Tau (1), P-Tau (2), pure recombinant hT40 (3), towhich had been added, just before spotting, the same amount of cytosolas used to generate P-Tau (4) or HP-Tau (5). Dot 6 refers to the GST (5μg) load.

FIGS. 4A-B: Colocalization of FKBP52 and TAU in primary cortical neuronsand PC12 cells. A) Immunofluorescence staining of primary corticalneurons and PC12 cells treated with 50 nM NGF for 5 days. Doublestaining for Tau and FKBP52 was performed after cytosol extraction toreveal cytoskeletal association. Arrows indicate preferentialcolocalization of both proteins in the distal part of the nerve cellaxon and at the extremity of PC12 cell neurites.

B) Confocal images of primary cortical neurons. Double staining wasperformed as in (A). Analysis of 0.5 μm slices confirms the preferentialcolocalization in the distal part of the axon (see arrowheads)

FIGS. 5A-G: Effect of FKBP52 on tubulin polymerization induced byrecombinant Tau isoforms. Tubulin polymerization was performed byswitching the samples from 4° C. to 37° C. and the change in turbiditywas monitored at 345 nm for 15 min. Tubulin (1 mg/ml) purified from ratbrain was incubated in the absence (♦) or presence of 1.7 μM (23 μg forHT40) different human Tau isoforms without FKBP52 (⋄) or with 3.5 μM (55μg) FKBP52 (▴) Tau isoforms differ from each other by the number ofrepeats in the microtubule binding domain and insertions in theN-terminal. The labelling of tau isoforms uses the publishednomenclature (28). A: ht40, B: ht39, C: hT37, D: hT34, E: hT24, F: hT23,G: This control experiment was carried out as in (A), except that 3.5 μMGST (▴) was used instead of FKBP52.

FIGS. 6A-F: Effect of FKBP52 overexpression on Tau accumulation andneurite outgrowth A) PC12 cells treated or not with NGF (50 nM) in theabsence or presence of 1 μg/ml Dox (doxycycline). Ten μg of totalprotein extracts were analyzed for FKBP52 levels by western blot usinganti-FKBP52 antibody 761. B) The FKBP52 level was determined as in (A)in H7C2 cells treated or not with Dox. The use of rabbit FKBP52 as theexogenous protein explains the small difference in gel mobility with theendogenous rat protein. C) H7C2 and D) PC12 cells were treated or notwith NGF for 5 days, in the presence or absence of Dox for one week; 50μg of extracts was subjected to SDS-PAGE and immunoblotted with anti-Tau(antibody clone DC25). Actin was used as the loading control. E)Representative H7C2 cells in the presence of NGF (50 nM) with or withoutDox. F) Neurite length was quantified from random photographs (seematerials and methods). Similar results were obtained in 3 separateexperiments. **: P<0.01 (Student-Newman-Keuls test, Anova).

FIGS. 7A-B: centrifugal sedimentation and western blot analysis of Taupolymerization: (A) in oxidative conditions: Ox (B) in reducingconditions: Red. S, Supernatants, P, Pellets.

FIG. 8: Centrifugal sedimentation and western blot analysis of Taupolymerization in oxidative conditions: recombinant Tau at 0.5 mg/ml 1:alone, 2: with FKBP52, 3: with GST. S, Supernatants, P, Pellets.

FIGS. 9A-F: Expression level of FKBP52 in Alzheimer disease and controlbrains. Western blot analysis of FKBP52 in soluble homogenate fractionfrom normal and Alzheimer disease. GAPDH was used as loading control.Intensities of the chemoluminescence were quantified with Image-Jsoftware. LA), soluble fractions 1-5; (B), soluble fractions 6-12; (C)soluble data combined; (D) insoluble fractions 1-5; (E), insolublefractions 6-12; (F) insoluble data combined.

FIGS. 10A-B: Expression level of mRNA FKBP52 in Alzheimer disease andcontrols. Quantification of mRNA FKBP52 was carried out afternormalisation with mRNA GAPDH. (A) individual samples; (B) combineddata.

EXAMPLE 1: A ROLE FOR FKBP52 IN TAU PROTEIN FUNCTION

Materials and Methods

Antibodies and Reagents:

Anti-Tau mAB (clone DC25) and anti-Tau mAB (Tau5) was from Sigma andCalbiochem respectively. Anti-FKBP52 pAB 761 was as described (9). GTPwas from Sigma and doxycycline was from Clontech.

Preparation of Tubulin and Microtubule Assembly Assay:

Male adult Sprague-Dawley rats (body weight˜250 g) were obtained fromJanvier (Le Genest-St.-Isle, France). They were killed by decapitation,according to institutional guidelines, and whole brains were usedimmediately to prepare tubulin as described (9). Microtubule assemblyassays were performed as in (9).

Protein Purification and Overexpression of Different Tau Isoforms andFKBP52 Protein:

The six isoforms of human brain Tau were expressed in E. coli fromclones hT40, hT39, hT37, hT34, hT24, hT23 and purified as described(28). Full length FKBP52 was affinity purified as in (23). For theTubulin polymerization assay, FKBP52 bound to glutathione-sepharosebeads (GE Healthcare) were cleaved overnight at 4° C. with 2 units ofthrombin (GE Healthcare) and dialyzed against buffer L (0.1 M Mes, 1 mMEGTA, 1 mM MgCL₂, 0.1 mM EDTA) with 10% glycerol, and complementedbefore use to 1 mM GTP and 1 mM DTT and 10 μM PPACK (a potentirreversible inhibitor of thrombin) (Biomol).

Phosphorylation and Hyperphosphorylation of Recombinant Tau:

Rat brain extract was used as the source of kinase activity, asdescribed (18). Briefly, recombinant hT40 was incubated with cytosol ofadult rat brain, in the presence or absence of okadaic acid, to giveHP-Tau (hyperphosphorylated-Tau) and P-Tau (phosphorylated-Tau),respectively.

Protein Binding Assays:

GST-pull down assay: 100 μl of glutathione-Sepharose beads preloadedwith 1 nmol GST-FKBP52 or 1 nmol GST, were washed 4 times with 500 μl ofbuffer A (buffer L complemented with 1 mM DTT and 1 mM GTP) and thenresuspended in the same buffer containing protein microtubule extract.The proteins were analyzed for the presence of Tau isoforms by SDS/PAGEwestern blot analysis using antibody anti-Tau (clone DC25) diluted1/1000.

Coimmunoprecipitation Assay:

It was carried out with 1 mg cytosol microtubule extract as described(12).

Dot Blot Assay:

100 μl of buffer A containing different amounts of hT40 were applied toa nitrocellulose membrane, blocked with 5% non-fat milk inphosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) at roomtemperature, washed with PBS-T and buffer A, followed by 2 h incubationat room temperature with 100 μl of buffer A containing 0.5 μgrecombinant FKBP52. The membranes were washed with buffer IP (50 mM Tris(pH 7.5)/150 mM NaCl/2 mM MgCl₂/0.1% Brij97 (Sigma)/10%glycerol/protease inhibitors) and with PBS-T. After blocking with milkin PBS-T, the membranes were incubated with anti-FKBP52 761 antibody.The presence of FKBP52 was revealed by ECL. Quantitation was performedwith Quantity-one software using Chemidoc XRS fitted with a 16 bit CCDcamera (Biorad).

Cell Line and Stable DNA Transfection:

Generation of H7C2 Cells:

The cDNA encoding rabbit FKBP52 was inserted into the HindIII and AccIrestriction sites of the pTRE2 vector (Clontech) in order to givepTRE2-FKBP52. Transfection of 100 μg of pTR2-FKBP52 and 10 μg ofpTK-hygromicin was carried out in a commercially available PC12 Tet-oncell line (Clontech) that expresses the reverse tetracycline controlledtransactivator, using Lipofectamine™ (Invitrogen). Stably transfectedcells were selected with 100 μg/ml hygromycin and screened individually.

Cell Culture:

PC12 cells and H7C2 cells were grown in DMEM containing 10% (v/v) horseserum and 5% (v/v) FBS (Invitrogen) at 37° C. in 90% 02/10% CO₂. Thedifferentiated neuronal phenotype of cells grown on plastic dishescoated with 10 μg/ml poly(L)-lysine (Sigma), was induced by adding nervegrowth factors (NGF) (Invitrogen) for 5 days. Primary cultures fromcerebral cortex of embryonic day 17 rat foetuses were carried out.Dissociated cells were plated (50,000 cells per ml) on glass coverslipscoated with poly(L-ornithine) and cultured in a defined medium in 95%O₂/5% CO₂ at 37° C.

Immunocytochemistry:

Cells were grown on glass coverslips precoated in 12-well tissue cultureplates. Primary cells and PC12 cells were incubated for 2.5 min and 3min respectively, in PEM buffer (80 mM PIPES, 1 mM MgCl₂, 2 mM EGTA,pH6.9) with 0.05% Triton, rinsed with warm Triton-free PEM, fixed for 5min with methanol at −20° C. and incubated with affinity-purifiedanti-FKBP52 761 (1/1000), and anti-Tau5 (1/100). Anti-rabbit Alexa Fluor488-conjugated (Invitrogen), anti-mouse FITC-conjugated or Cy™3red-conjugated (GE-Healthcare) antibodies were used at 1/500 and 1/1000,respectively. The coverslips were examined by epifluorescence using aZeiss axioplan 2 microscope with, either a ×63 objective or by confocalmicroscopy (Zeiss, Thornwood, N.Y., USA)

Quantitation of Neurite Outgrowth:

Random field photographs of PC12 and H7C2 cells, in each of three wells,were analyzed with Neuron J software. The average neurite length wasdetermined by measuring the longest neurites of at least 200 cellsrandomly selected.

Results

Tau FKBP52 Association:

FKBP52 is widely distributed in the brain as shown by Western blots ofcytosolic proteins from several brain areas (FIG. 1). In order Toinvestigate whether MAPs may be involved in the effect of FKBP52 onmicrotubule stability (9), GST pull-down assays were carried outincubating GST-FKBP52 bound to sepharose beads with microtubules cytosolprepared from adult rat brain. Specifically bound proteins were analyzedby immunoblotting using antibodies directed against MAP1b, MAP2 and Tau.In these experimental conditions, no immunoreactivity was observed forMAP1b or MAP2, but Tau immunoreactivity was present (FIG. 2A). In ratbrain homogenates, Tau appears as multiple bands representing differentsplice isoforms with various degrees of phosphorylation. Several Tauspecies were also found in pull-down experiments of rat brain cytosolmicrotubules using GST-FKBP52. Tau immunoreactivity was not detected incontrols using purified GST (FIG. 2A). To confirm the specificity ofthis association, microtubules of adult rat brain cytosol wereimmunoprecipitated with a polyclonal antibody against FKBP52.Immunoprecipitates were analyzed by Western blotting with a monoclonalTau antibody. Tau co-immunoprecipitated with FKBP52 but not with apreimmune serum (FIG. 2B). Thus, Tau and FKBP52 form a complex in ratbrain. These experiments do not address whether the binding of Tau toFKBP52 is direct or whether it involves additional factors. Toinvestigate this, recombinant Tau (hT40, the longest isoform, expressedin E. coli and purified) was spotted onto nitrocellulose and incubatedwith purified recombinant FKBP52. Then, proteins sequestered by Tau weredetected with a polyclonal antibody against FKBP52. As shown on FIG. 2C,FKBP52 was retained in a dose dependent manner by Tau, but not by theGST. These findings indicate a direct interaction between FKBP52 andTau.

Effect of Tau Phosphorylation on its Interaction with FKBP52:

To define whether the phosphorylation of Tau modulates its associationwith FKBP52, dot blot experiments were performed using recombinantphosphorylated Tau (P-Tau) and hyperphosphorylated Tau (HP-Tau) (18)(FIG. 3A). The specificity of the interaction between FKBP52 and P-Tauor HP-Tau was determined by experiments using as the bait either thesephosphorylated ht40, non phosphorylated hT40, or hT40 to which had beenadded, just prior to spotting, the same amount of cytosol protein asused to obtain phosphorylated or hyperphosphorylated hT40. As shown inFIG. 3B, the amount of FKBP52 recruited by Tau depends on itsphosphorylation state. 73% (±7) of FKBP52 was retained by 2.2 μg HP-Tau,whereas only 3.5% and 6.6% were respectively retained by the same amountof hT40 and by hT40 in the presence of cytosolic proteins used ascontrols. When P-Tau was used as bait, only 41% (±15) of FKBP52 wascaptured. This difference in binding between HP-Tau and P-Tau to FKBP52may be explained by the different degree of Tau phosphorylation atspecific sites or by the global amount of Tau phosphorylation, or by acombination of both mechanisms (FIG. 3A). In any case, these resultsunderline the importance of Tau phosphorylation for its binding toFKBP52.

Colocalization of Tau and FKBP52 in Primary Cortical Neurons and PC12Cells.

The subcellular localization of Tau and FKBP52 was examined byimmunofluorescence experiments with rat primary cortical neurons. After6 days of culture, and mild extraction selectively removing unboundcytosolic proteins while retaining proteins associated with thecytoskeleton, double staining was performed on neurons with monoclonalantibody Tau5 and affinity-purified polyclonal anti-FKBP52 antibody. Inagreement with an earlier report (19), Tau was concentrated in thedistal portion of axons and at the growth cone neck, where a strongaccumulation of FKBP52 was also observed (FIG. 4). Colocalization andaccumulation of FKBP52 and Tau were also found at the growth cones ofPC12 cells (FIG. 4). Very recently it has been reported that Tau isselectively enriched at axonal tips and that this may be due to itsspecific anchoring (20). Our results suggest that FKBP52 may be involvedin the trapping of Tau, and thereby able to influence its subcellulardistribution.

FKBP52 Inhibits Tubulin Polymerization Induced by Tau In Vitro.

To demonstrate a functional interaction between Tau and FKBP52, amicrotubule kinetic assay was set up. In experiments with purified ratbrain tubulin alone, no microtubule was formed, whereas in experimentswith recombinant human Tau isoforms, an increased absorbance reflectingmicrotubule assembly was observed (FIG. 5). However, when Tau was addedto the tubulin in the presence of purified recombinant FKBP52, formationof microtubules was prevented, whereas GST was ineffective. Similarresults were obtained with the 6 isoforms of human Tau (FIG. 5). Weconcluded that FKBP52 inhibits the promotion of microtubule assembly byTau.

FKBP52 Prevents Tau Accumulation and Neurite Outgrowth in PC12 Cells:

An FKBP52-inducible expression system based on a tetracycline-responsiveelement allowing the generation of a stably transformed PC12 cell linewas used (21) to determine a cellular role for FKBP52. Among cloneswhich were positively tested, one clone, so called H7C2, was selectedand used to study the effects of FKBP52 overexpression on PC12 cells,and to further investigate the possible relationship between FKBP52 andTau. Under basal conditions H7C2 cells expressed endogenous FKBP52, andtreatment with Dox (doxycycline) resulted in a marked increase ofrecombinant FKBP52 protein expression (FIG. 6A). FKBP52 induction inH7C2 cells was about 4 fold after 5 days of Dox treatment.

The effect of FKBP52 on the accumulation of Tau was examined next. Theamount of Tau protein was determined by Western blotting of extractsfrom cultures of either PC12 cells or H7C2 cells, treated or not withNGF (50 nM) for 5 days with or without Dox. In PC12 cells, FKBP52expression was unchanged after treatment with NGF (FIG. 6B). Asexpected, in both PC12 and H7C2 cells an increase in Tau was observedafter NGF treatment. However, when H7C2 cells were exposed to Dox inaddition to NGF, thus overexpressing FKBP52, no additional accumulationof Tau protein occurred. An increase in Tau protein was still observedin PC12 cells treated with NGF and Dox, ruling out the possibility thatDox was responsible for the lack of decrease in Tau (FIG. 6C). Inconclusion, FKBP52 prevented the accumulation of Tau induced by NGF inPC12 cells.

Since one role of Tau is to stimulate neurite outgrowth (12), weinvestigated the consequence of FKBP52 overexpression on neurite lengthin both PC12 and H7C2 cells. In the absence of NGF, no neurite outgrowthwas observed in H7C2 cells, whether or not they were treated with Doxfor a week. However in H7C2 cells treated with 50 nM NGF and Dox, a 40%(+7) decrease in neurite length, compared to control (H7C2 not treatedwith Dox) was observed (FIG. 6D). The same effect of Dox on neuritelength was observed in H7C2 cells treated with 10 or 20 nM NGF. That Doxby itself was involved in the process of neurite outgrowth could beruled out, since no difference in neurite length between Dox-treated anduntreated PC12 cells was observed. The inhibition of neurite outgrowthresulting from FKBP52 overexpression is in agreement with our previousreport showing that the loss of FKBP52 in PC12 cells results in theformation of neurite extensions (9). The FKBP52 effect on neurite lengthcould be explained by the binding of Tau to FKBP52, removing Tau frommicrotubules. In addition the prevention of Tau accumulation byoverexpression of FKBP52 is consistent with the decrease of neuritelength and evokes a potential role of this immunophilin in Tau function.

Discussion

This newly discovered “anti-Tau” activity of FKBP52 leads us tore-examine the functions of this protein, which was originallyidentified and cloned as modulator of hormone steroid receptors (8, 22).FKPB52 is a multimodular protein, which includes a peptidyl prolylisomerase (“rotamase”) segment, the function of which is blocked byFK506 (23), rapamycin and some related non-immunosuppressivederivatives. There is a noteworthy structural similarity between FKBP52and Pin1: both proteins have a peptidyl-prolyl isomerase (PPIase)activity and a specific protein-protein interaction domain (7). Sincethe Pin1 PPIase activity restores the function of phosphorylated Tauprotein in a model of Alzheimer's disease (7), the interaction observedbetween Tau and FKBP52 may have implications for the pathogenesis of thetauopathies, including Alzheimer's disease. It must be remembered that,unlike FKBP12 (24) FKBP52 does not bind calcineurin (25), and thusFKBP52 does not mediate the immunosuppressant capacity of FK506.Therefore, the pharmacological modulation of the rotamase activity ofFKBP52 by non-immunosuppressive FK506/rapamycin derivatives may offer anovel approach for preventing/reducing the pathogenic effects ofmisfolded Tau.

In addition to its peptidyl prolyl isomerase activity, FKBP52 serves asa molecular chaperone. This activity depends on its tetratricopeptiderepeat domain (26) to which the molecular chaperone HSP90 and otherproteins bind. It has already been noted that chaperone co-chaperoneprotein complexes play a critical role in neurodegenerative diseasescharacterized by Tau accumulation (27). We now report that FKBP52 coulddecrease the function/accumulation of Tau, and therefore suggest itspossible involvement in these described cochaperone systems (27).

Our results establish a role of FKBP52 in Tau function. The interactiondescribed in this report rapidly deserves to be studied further, sinceeffective pharmacological targeting of FKBP52 is likely to become areality in the near future.

EXAMPLE 2: EFFECT OF FKBP52 ON OLIGOMERIZATION OF TAU IN VITRO

Aggregates of Tau protein are characteristic of multipleneurodegenerative diseases. (Delacourte A and Bué L, 2000). Studies ofconditions for Tau aggregation in vitro have led to differentexperimental systems including oxidative conditions, inductions bypolyanions such as heparin, and by fatty acids such as arachidonic acid(Barghorn S and Mandelkow E, 2002).

To investigate the effect of FKBP52 on Tau oligomerization we monitoredby sedimentation assay the ability of recombinant Tau protein (hereusing the longest isoform) to polymerise in oxidative conditions inpresence or absence of recombinant FKBP52. After dialysis of Tau (0.5mg/ml) overnight at 4° C. in Tris 10 mM pH7.4 and centrifugation at14000 rpm, quantitative western blotting was used to monitor thepolymerization of Tau protein. In this condition sedimentation analysisrevealed the presence of Tau in pellet fraction contrary to theexperiment carried out in presence of DTT where no detectable pelletablepolymers of Tau could be obtained (FIG. 7). This experiment means thatTau in oxidative conditions self polymerise and confirms, as alreadyreported, the importance of cysteines in first step of Tauoligomerization process (Schweers et al 1995).

The effects of coincubating, in oxidative medium, Tau and FKBP52 on Tauoligomerization process were monitored. As shown on FIG. 8 no detectablepresence of Tau in pellet fraction could be observed when dialysis wereperformed in equimolecular presence of FKBP52 where a pelletablepolymers of Tau could be observed when coincubation was carried out inpresence of GST used as control.

However when sedimentation analysis experiments were realized in varyingthe concentration of FKBP52 (1 to 0.1 mg/ml) where the concentration ofTau was constant (0.5 mg/ml) the presence of FKBP52 at 0.1 or at 0.25mg/ml results in increasing Tau polymers in pellet where the presence ofequimolecular or two times quantities of FKBP52 versus Tau prevent thepresence of pelletable polymers of Tau (FIG. 9). These results suggest adual role for FKBP52: firstly: a FKBP52 conformational activity on Tau,possibly involving peptidyl prolyl isomerase activity of FKBP52, leadingTau in a favourable proaggregative form; secondly: FKBP52 could protecteby possible steric hindrance the ability of Tau to oligomerize via theformation of disulfide bridges.

EXAMPLE 3: DOWN REGULATION OF FKBP52 PROTEIN IN BRAINS FROM PATIENTSWITH ALZHEIMER DISEASE

Human brain pre frontal cortex, obtained thanks to the access ofNational BrainBank (GIE NeuroCEB), were homogenized in 5 volumes ofbuffer containing Tris 10 mM, Saccharose 0.32M, DTT 1 mM and proteaseinhibitor cocktail. The homogenates were centrifugated at 14000 RPM at4° C. for 5 min. The supernatant was used as soluble fraction. Thepellet was solubilised by homogenization in the same conditions and usedas insoluble fraction. FKBP52 levels in the soluble and insolublefractions were analyzed by western blotting. The FKBP52 level in solubleand insoluble fraction represents respectively 80% and 20% of totalFKBP52;

In the aim of elucidate if FKBP52 protein expression level might bealtered in patient brain we have compared its expression in the prefrontal cortex of normal control (n=3) and patients with Alzheimerdisease (n=12). As shown on FIG. 10 marked reduction of FKBP52 inAlzheimer disease brains is observed in soluble fraction. Afternormalization with GAPDH (Glyceraldehyde 3-phosphatedehydrogenase)expression levels of FKBP52 found in soluble fraction of Alzheimerdisease brains represent 35% (±24) compared to the controls. Nomodification of expression protein level of FKBP52 could be detectablein insoluble fraction; this last observation suggests that FKBP52 is nottrapped in aggregates of Tau.

Subsequently the mRNA expression level of FKBP52 gene was analyzed byreal time quantitative RT-PCR (QPCR) in prefrontal cortex of human braindisease and controls. As shown on FIG. 11 no modification of FKBP52 mRNAlevel is observed in brain diseases versus control. These resultsindicate that the decreasing of FKBP52 protein expression in the patientbrain is due to a post transcriptional mechanism.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

-   Ballatore C, Lee V M Y, Trojanowski J Q (2007) Tau-mediated    neurodegeneration in Alzheimer's disease and related disorders. Nat    Rev Neurosci 8: 663-672.-   Barghorn S and Mandelkow E. Toward a unified scheme for the    aggregation of tau into Alzheimer paired helical filaments.    Biochemistry. (2002); 41:14885-96.-   Buée L, Bussière T, Buée-Scherrer V, Delacourte A, Hof P R (2000)    Tau protein isoforms, phosphorylation and role in neurodegenerative    disorders. Brain Research Review 33: 95-130.-   Chambraud B et al (1993) Overexpression of p59-HBI (FKBP52), full    length and domains, and characterization of PPIase activity.    Biochem. Biophys. Res. Commun. 196: 160-166.-   Chambraud B, Belabes H, Fontaine-Lenoir V, Fellous A, Baulieu E    E (2007) The immunophilin FKBP52 specifically binds to tubulin and    prevents microtubule formation. FASEB. J 11: 2787-2797.-   Delacourte A and Buée L. Tau pathology: a marker of    neurodegenerative disorders. Curr Opin neurology, (2000) 13:    371-376.-   Delacourte A, Buée L (2000) Tau pathology: a marker of    neurodegenerative disorders. Curr Opin neurology 13: 371-376.-   Dickey C et al (2007) The high-affinity HSP90-CHIP complex    recognizes and selectively degrades phosphorylated tau client    proteins. J. Clin; Invest. 117: 648-658.-   Garcia M L, Cleveland D W (2001) Going new places using an old MAP:    tau, microtubules and human neurodegenerative disease. Curr Opin    Cell Biol 13: 41-48.-   Goedert M, Jakes R (1990) Expression of separate isoforms of human    tau protein:correlation with the tau pattern in brain and effects on    tubulin polymerization EMBO. J. 9: 4225-4230.-   Goedert M, Spillantini M G, Jakes R, Rutherford D, Crowther R    A (1989) Multiple isoforms of human microtubule-associated    protein-tau: sequences and localization in neurofibrillary tangles    of Alzheimer-disease. Neuron 3: 519-526.-   Goedert M, Wischick C M, Crowther R A, Walker J E, Klug A (1988)    Cloning and sequencing of the cDNA encoding a core protein of the    paired helical filament of Alzheimer disease: Identification as the    microtubule-associated protein tau. Proc Natl Acad Sci USA 85:    4051-4055.-   Goedert M. et al (1993) The abnormal phosphorylation of tau protein    at Ser-202 in Alzheimer disease recapitulates phosphorylation during    development. Proc. Natl. Acad. Sci. U.S.A. 90: 5066-5070.-   Gold B G (1997) FK506 and the Role of Immunophilins in Nerve    Regeneration. Mol Neurobiol 15: 285-306.-   Gossen M, Bujard H (1992) Tight control of gene expression in    mammalian cells by tetracycline-responsive promoters. Proc. Natl.    Acad Sci. U.S.A. 89: 5547-5551.-   Kempf M, Clement A, Faissner A, Lee G, Brandt R (1996) Tau Binds to    the Distal Axon Early in Development of Polarity in a Microtubule-    and Microfilament-Dependent Manner. J Neurosci 16:5583-5592.-   Lebeau M C et al (1992) P59, an hsp 90-binding Protein. J Biol Chem.    267, 4281-4284.-   Lebeau M C, Myagkikh I, Rouvière-Fourmy N, Baulieu E E, Klee C    B (1994) Rabbit FKBP-59/HBI does not inhibit calcineurin activity in    vitro. Biochem. Biophys. Res. Com. 203: 750-755.-   Lee V M Y, Goedert M, Trojanowski J Q (2001) Neurodegenerative    Tauopathies. Annu Rev Neurosci 24: 1121-1159.-   Liu J et al (1991). Calcineurin is a common target of    cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66:    807-815.-   Lu K. P, Zhou X. Z. (2007) The prolylisomerase PIN1: a pivotal new    twist in phosphorylation signalling and disease. Nature Rev Mol.    Cell. Biol 8: 904-916.-   Mandelkow E M, Mandelkow E (1998) Tau in Alzheimer's disease. Trends    in Cell Biology 8: 425-427.-   Pirkl F, Fischer E, Modrow S, Buchner J (2001) Localization of the    Chaperone Domain of FKBP52. J Biol. Chem 276: 37034-37041.-   Riggs D L et al (2003) The Hsp90-binding peptidyl isomerise FKBP52    potentiates glucocorticoid signalling in vivo. EMBO. J. 22:    1158-1167.-   Schiene C, Fischer G (2000) Enzymes that catalyse the restructuring    of proteins. Curr Opin Struct Biol 10: 40-45.-   Schreiber S L (1991) Chemistry and Biology of the Immunophilins and    Their Immunosuppressive Ligands. Science 251: 283-287.-   Schweers O, Mandelkow E-M, Biernat J, and Mandelkow E. Oxidation of    cysteine-322 in the repeat domain of microtubule-associated protein    tau controls the in vitro assembly of paired helical filaments. Proc    Natl Acad Sci. U.S.A. (1995). 92:8463-8467.-   Shim S et al (2009) Peptidyl-Prolyl Isomerase FKBP52 Controls    Chemotropic Guidance of Neuronal Growth Cones via Regulation of    TRPCl Channel Opening. Neuron 64: 471-483.-   Snyder S H, Sabatini D M (1995) Immunophilins and the nervous    system. Nat Med 1: 32-37.-   Steiner J P et al (1992) High brain densities of the immunophilin    FKBP colocalized with calcineurin. Nature 358: 584-587.-   Weissmann C et al (2009) Microtubule Binding and Trapping at the Tip    of Neurites Regulate Tau Motion in Living Neurons. Traffic    11:1655-1668

1-10. (canceled)
 11. A method of treating neurological disordersinvolving Tau dysfunction, in a subject thought to have or bepredisposed to having a neurological disorder involving Tau dysfunction,comprising the administration to said subject of an effective amount ofa compound which enhances the interaction between Tau and FKBP52proteins, so as to prevent aggregation of Tau protein.
 12. The methodaccording to claim 11, wherein the neurological disorders involving Taudysfunction are selected from the group consisting of Alzheimer'sdisease, frontotemporal dementia, progressive supranuclear palsy,corticobasal degeneration and frontotemporal lobar degeneration.
 13. Themethod according to claim 11, wherein said compound is combined withpharmaceutically acceptable excipients.
 14. The method according toclaim 11, wherein the compound is selected among candidate compounds bya screening method comprising the steps of: a) determining the abilityof the candidate compound to enhance the interaction between Tau andFKBP52 proteins, and b) selecting positively the candidate compound ifthe candidate compound enhances said interaction.
 15. A method oftreating Alzheimer disease comprising the administration to a subject inneed thereof of an therapeutically effective amount of a compound thatmodulate both the interaction between Tau and FKBP52 proteins and theinteraction between FKBP52 and APP proteins.